1. A Fructose Receptor Functions as a Nutrient Sensor in the Drosophila Brain 
Tetsuya Miyamoto, Jesse Slone, Xiangyu Song, Hubert Amrein 
Cell 
Volume 151, Issue 5, Pages (November 2012)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
Gr43aGAL4 Is Expressed in Chemosensory Organs, the Brain, and the Proventriculus
(A–C) Expression of Gr43a in chemosensory organs. Gr43aGAL4 drives strong UAS-mCD8GFP expression in neurons located in the fifth tarsal segment of the foreleg (A1, live GFP), the LSO and the VCSO (B1; live GFP), but only weak expression in the LPs (C1; immunostaining). Coexpression analysis in sweet neurons was performed in flies containing Gr43aGAL4 and Gr64fLexA, driving expression of UAS-mCD8RFP (detected with anti-CD8 antibody) and lexAop-rCD2GFP (detected with anti-GFP antibody), respectively. Coexpression analysis in bitter neurons was performed in flies containing Gr43aGAL4 driving expression of UAS-mCD8RFP (detected with anti-CD8 antibody) and Gr66a-gfp (detected with anti-GFP antibody). LP, labial palp; LSO, labral sensory organ; VCSO, ventral cibarial sense organ. Arrowheads indicate Gr43aGAL4 neurons.
(D) Gr43aGAL4 is expressed in two to four neurons/hemisphere in the posterior superior lateral protocerebrum.
(E) Gr43aGAL4 is expressed in approximately four neurons in the proventricular ganglion. Gr43aGAL4 neurons innervate the lumen of the foregut, but not the crop duct (E2, left inset; E1). Some neurons send projections to the mid gut (E2, right inset; E1, arrow; E3), others to the SOG (E1, arrowhead). Pv, proventriculus; Cr, crop; Mg, mid gut.
See also Figures S1, S2, and S3.
Cell  , DOI: ( /j.cell )
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4. Figure 2 Gr43a Is a Fructose Receptor
(A) Tarsal neuron expressing G-CaMP3.0 under control of Gr43aGAL4: ΔF pseudocolor fluorescence image was taken 1.5 s after application of 100 mM fructose (right).
(B) G-CaMP3.0 (ΔF/F) fluorescence is dose-dependent (fructose was used as ligand).
(C) Gr43aGAL4 neurons responded to sugars, but not to bitter compounds, acid, and base. Max ΔF/F within 30 s of applications is shown. Sugars and caffeine were at 100 mM, quinine and denatonium at 10 mM concentration. Error bars represent SE (3 ≤ n ≤ 8).
(D) Ca2+ response of Gr43aGAL4 neurons with/without Gr43a, Gr61a and Gr64a-f to various sugars. From left to right, genotypes are Gr43aGAL4/+, Gr43aGAL4/Gr43aGAL4, Gr43aGAL4/Gr43aGAL4;UAS-Gr43a, Gr43aGAL4/+;ΔGr61a ΔGr64a-f/ΔGr61a ΔGr64a-f, Gr43aGAL4/Gr43aGAL4;ΔGr61a ΔGr64a-f/ΔGr61a ΔGr64a-f, Gr43aGAL4/Gr43aGAL4;UAS-Gr43aΔGr61a ΔGr64a-f/ΔGr61a ΔGr64a-f. All sugars are 100 mM. NS, not significant; ∗p < 0.05; ∗∗p < ; ANOVA. Error bars represent SE (8 ≤ n ≤ 9).
(E) Gr43a is sufficient to induce PER response to fructose. All sugars are 100 mM. NS, not significant; ∗p < 0.05; ANOVA. Error bars represent SE (9 ≤ n ≤ 10 experiments; 9–21 flies per experiment).
See also Movie S1.
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5. Figure 3 Gr43a Functions as a Fructose Sensor in the Brain
(A) Brain neuron expressing G-CaMP3.0 under control of Gr43aGAL4: ΔF pseudocolor fluorescence image was taken 24 s after application of 100 mM fructose (right).
(B) Gr43aGAL4 neurons specifically respond to fructose. Max ΔF/F within 15 min of application is shown. All sugars are 100 mM. Flies contained two genomic copies of Gr43a. ∗∗p < ; ANOVA. Error bars represent SE (6 ≤ n ≤ 7).
(C) Response of Gr43aGAL4 neurons to fructose is dose- and Gr43a-dependent. ∗∗p < ; ANOVA. Error bars represent SE (8 ≤ n ≤ 9).
(D) Time-course of G-CaMP3.0 fluorescence changes in Gr43aGAL4 neurons stimulated with different concentrations of fructose.
See also Movie S2.
Cell  , DOI: ( /j.cell )
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6. Figure 4 Metabolic Dynamics of Circulating Sugars
Flies were starved for 24 hr (pre), followed by 40 min of feeding. Measurements were performed at indicated times after feeding (see Experimental Procedures).
(A) Relative change of internal glucose, trehalose, and fructose over time.
(B) Amount of glucose, trehalose, and fructose (per mg of head tissue) was measured immediately after feeding. ∗p < 0.05; ANOVA. 5 ≤ n ≤ 12. Last column (#) shows concentration of fructose (converted to mM) using the conservative estimate that one-fifth of the insect mass constitutes hemolymph (Chapman, 1998).
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7. Figure 5 Gr43a Functions as an Internal Nutrient Sensor
(A) Gr43a evaluates nutritional content of carbohydrates. Single flies were subjected to the CAFÉ assay by presenting them with two capillaries containing water and 100 mM sorbitol, respectively, for 24 hr. NS, not significant; ∗p < 0.05; ∗∗p < 0.01; ANOVA. Error bars represent SE (50 ≤ n ≤ 74).
(B) Cha7.4kb-GAL80 restricts Gr43aGAL4 expression to the brain neurons. GFP expression (arrowhead) is missing in all sensory neurons, but remains robust in the brain neurons. Likewise, all projections of sensory neurons to the SOG (arrow) disappeared.
(C) Average number of Gr43aGAL4 neurons in different tissues. Error bars represent SE. 3 ≤ n ≤ 11 (without Cha7.4kb-GAL80), 6 ≤ n ≤ 15 (with Cha7.4kb-GAL80). Note that a few legs of flies with Cha7.4kb-GAL80 show very weak GFP expressions.
(D) Gr43aGAL4 brain neurons are necessary to evaluate nutritional content of carbohydrates. Flies with silenced Gr43aGAL4 brain neurons (Gr43aGAL4/UAS-TNT; Cha7.4kb-GAL80) lack sorbitol preference, in contrast to control flies. ∗p < 0.05; ANOVA. Error bars represent SE (64 ≤ n ≤ 83).
See also Figure S4.
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8. Figure 6
Gr43a Function in Brain Neurons Suppresses Feeding in Satiated Flies and Promote Feeding in Hungry Flies
Single flies were subjected to the CAFÉ assay for 24 hr by presenting them a single capillary containing the indicated solution.
(A) Gr43a suppresses nutritious sugar consumption under satiated conditions. NS, not significant; ∗p < 0.05; ∗∗p < 0.01; ANOVA. Error bars represent SE (21 ≤ n ≤ 34). All sugars were used at 100 mM concentration, except sucrose, which was used at 50 mM, to obtain equal nutritional value for mono- and disaccharides.
(B) Nonnutritious sugars arabinose (100 mM), xylose (100 mM), and sucralose (50 mM) were consumed in equal amounts by control and Gr43aGAL4 mutant flies. NS, not significant; ANOVA. Error bars represent SE (24 ≤ n ≤ 27).
(C) Gr43aGAL4 brain neurons are sufficient to suppress and promote feeding in satiated and hungry flies, respectively. Cha7.4kb-GAL80 restricts expression of Gr43aGAL4 to the brain (see Figures 5B and 5C). Sucralose (50 mM) was added to “sweeten” sorbitol (100 mM), enhance feeding, and achieve satiation. For hungry state, sorbitol (100 mM) alone was used. NS, not significant; ∗p < 0.05; ANOVA. Error bars represent SE (35 ≤ n ≤ 84).
(D) Gr43aGAL4 brain neurons are necessary to suppress and promote feeding in satiated and hungry flies, respectively. Flies with silenced Gr43aGAL4 brain neurons (Gr43aGAL4/UAS-TNT; Cha7.4kb-GAL80) consume more nutritious food (sucralose + sorbitol) under satiating conditions, but less under nonsatiating conditions (sorbitol alone). NS, not significant; ∗p < 0.05; ANOVA. Error bars represent SE (41 ≤ n ≤ 88).
Cell  , DOI: ( /j.cell )
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9. Figure 7
Activation of Gr43aGAL4 Brain Neurons Assigns Satiety-Dependent Valence
(A) Schematic diagram of the olfactory conditioning assay. Flies are exposed to odor A, whereas their Gr43aGAL4 brain neurons are activated using trpA1 (29°C). After a brief rest period in an odorless vial, they are exposed to odor B in the absence of neural activation (23°C), followed by another rest period in an odorless vial. This training session is repeated once more before the flies are tested in a T-maze assay for acquired odor preference.
(B) Olfactory conditioning assay of starved and satiated flies. Prior conditioning, flies were kept in agarose or agarose containing 250 mM sucrose for 18–24 hr to induce starvation and satiation, respectively. Gr43aGAL4/UAS-trpA1;Cha7.4kb-GAL80/+ flies assign positive valence when hungry (pleasant; left graph), but negative valence when satiated (unpleasant; right graph). Control flies were Gr43aGAL4/+; Cha7.4kb-GAL80/+, and UAS-trpA1/+. ∗p < 0.05; ∗∗p < 0.01; ANOVA. Error bars represent SE (12 ≤ n ≤ 18).
(C) Model: the fly’s major blood sugars, glucose, and trehalose are kept at a fairly constant, relatively high level. Conversely, internal fructose level is very low, but fluctuates in response to feeding of nutritious sugars. Because nutritious carbohydrates can be converted into fructose, the activity of Gr43aGAL4 brain neurons depends on the nutritious value of the ingested food. Activation of Gr43aGAL4 brain neurons, in combination with the state of satiety, leads either to a pleasant sensation in hungry flies, reinforcing feeding behavior, or is perceived as unpleasant, thereby terminating feeding behavior.
See also Figure S5.
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10. Figure S1 Generation of the Gr43aGAL4 Allele, Related to Figure 1
(A) Organization of the wild-type and the GAL4-targeted Gr43a locus. The Gr43a coding region was replaced with GAL4 and whitemini by homologous recombination. The whitemini gene was removed via flanking loxP sites using CRE recombinase.
(B) Southern and PCR analysis of Gr43a+ and Gr43aGAL4 allele using probes and PCR primers indicated in (A). Genomic DNA was digested with SacI (S; 5′ probe) and XhoI (X; 3′ probe).
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11. Figure S2
Characterization of Gr43aGAL4 Proventricular Ganglion, Related to Figure 1
(A) Some projections of Gr43aGAL4 neurons pass through the brain along the esophagus and make a turn at the intersection of the proboscis and head capsule, where they join the labral nerve and enter the SOG (inlet, arrowhead).
(B) The presynaptic marker syt-HA is localized in projections to the midgut.
(C) The dendritic marker DenMark is enriched in innervations of the foregut lumen. Br, brain; Pb, proboscis; Es, esophagus; SOG, subesophageal ganglion; Fg, foregut; Cd, crop duct; Pv, proventriculus.
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12. Figure S3 Coexpression of Gr43aGAL4 and Gr5alexA, Related to Figure 2
(A) The distal Gr43aGAL4 neurons (arrowhead), but not the central neurons used for Ca2+ imaging, co-express Gr5alexA in the foreleg.
(B) Some neurons co-express Gr43aGAL4 and Gr5alexA in the labial palp (arrowhead).
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13. Figure S4 Sorbitol is Tasteless, Related to Figure 5
(A) Probability of proboscis extension reflex (PER) upon stimulation of the foreleg with water, sorbitol (100 mM) or fructose (100 mM). Sorbitol failed to elicit PER. NS, not significant; ∗p < 0.05; ANOVA. Error bars represent standard error. n = 8 experiments (4-9 flies per experiments).
(B) Ca2+ imaging of Gr43aGAL4 neurons in the forelegs when stimulated with water, sorbitol (100 mM) or fructose (100 mM). Gr43aGAL4 neurons do not respond to sorbitol. NS, not significant; ∗∗p < ; ANOVA. Error bars represent standard error. 9 ≤ n ≤ 11.
Cell  , DOI: ( /j.cell )
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14. Figure S5
Distinct Projections of the Gr43aGAL4 Brain Neurons, Related to Figure 7
(A) Diagram of the projection pattern of the Gr43aGAL4 brain neurons. Solid and dotted lines represent projections to the anterior and posterior part of the brain, respectively. All cell bodies are located in the posterior superior protocerebrum, and project axons to the middle inferior lateral protocerebrum (anterior projections) and to the posterior superior protocerebrum (posterior projections).
(B) Images of Gr43aGAL4 neuronal projections. Gr43aGAL4 was used to express mCD8GFP (membrane marker, green) and syt-HA (presynaptic marker, red). The Gr43aGAL4 brain neurons show distinct projection pattern. Some axons project toward the anterior brain along the middle inferior lateral protocerebrum. Other neurons project to the posterior superior lateral and medial protocerebrum, and few axons cross the midline and extend into the opposite side of the brain. Both anterior and posterior projections have numerous axon terminals.
Cell  , DOI: ( /j.cell )
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